The Establishment And Application Of Detection Methods For Pentatrichomonas Hominis

Pentatrichomonas hominis is a kind of opportunistic pathogenic zoonotic parasite, hasing abundant host animals and usually living in the cecum and colon. In recent years, there are persons who have been infected by pentatrichomonas hominis in Henan, Hubei, Guangdong, Anhui and other provinces, what is more, researchers have found pentatrichomonas hominis in the fecal samples of dogs, cats, non primate animals and rodent animals. In order to explore the pentatrichomonas hominis infection in deers and further assess the zoonotic potential, it is very important to build a fast and effective detection method to evaluate the pentatrichomonas hominis infection. Common intestinal Trichomonas detection methods include direct smear of microscopic examination, culture method, staining microscopy, conventional PCR detection method, nested PCR and hybridization detection method etc., however, direct smear of microscopic examination, culture method, conventional PCR assay and staining microscopic are not highly accuracy methods, hybridization need high requirements and complex procedures of experimental conditions, while nested PCR method for detection is often regarded as a quick, convenient, and effective detection method. We designed special primers of 18 S r RNA for nested PCR and then optimize the magnesium ion concentration and annealing temperature, results showed that the optimal concentration of magnesium ion was 2.5mmol/L and the optimal annealing temperature of two rounds of PCR reaction were 53 degree. Special test of the built method showed no positive reaction of genomic DNA of Eimeria tenella, Trypanosoma evansi, Toxoplasma gondii, Trichomonas vaginalis and Giardia lamblia, sensitive test showed that the method can test the minimum of 10 pentatrichomonas hominis/ml.The results of the 139 deer fecel samples showed 21.59%(30/139) pentatrichomonas hominis infection and 54.68%(76/139) tetratrichomonas buttreyi infection. What is more, we also designed special primers of EF-1α for nested PCR and then optimize the magnesium ion concentration and annealing temperature, results showed that the optimal concentration of magnesium ion was 0.75mmol/L and the optimal annealing temperature of two rounds of PCR reaction were 57 degree and 55 degree respectly. Special test of the built method showed no positive reaction of genomic DNA of Eimeria tenella, Trypanosoma evansi, Toxoplasma gondii, Trichomonas vaginalis and Giardia lamblia, sensitive test showed that the method can test the minimum of 10 pentatrichomonas hominis/ml. The results of the 139 deer fecel samples showed 21.59%(30/139). Sequences assay of these positive samples which tested by two nested methods, results showed that the mutation sites of detected pentatrichomonas hominis positive samples of 18 S r RNA gene sequence were mainly concentrated in three parts: the 1034 base differences in C/T, the 1051-1053 bases differences of TGC/TGT/GAT and the 1144-1165 bases exist complex diversity. However, detected pentatrichomonas hominis positive samples of EF-1α gene sequences showed no polymorphism. In conclusion, the nested PCR method based on EF-1α gene is more suitable for the detection of pentatrichomonas hominis infection and nested PCR detection method based on 18 S r RNA gene is more suitable for gene subtype identification of pentatrichomonas hominis. In the process of clinical detection, nested PCR method based on EF-1α gene should be used first for detection and then nested PCR detection method based on 18 S r RNA gene should be used to detect the positive samples for gene subtype identification assay. Our research successfully constructed the diagnosis methods based on specificity diagnosis genes and provided new methods for the study of epidemiology.